Dresden 2026 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 9: Single Molecule Biophysics

BP 9.1: Hauptvortrag

Montag, 9. März 2026, 16:45–17:15, BAR/0205

Breaking the photobleaching limit in single-molecule FRET with nanophotonic DyeCycling. — Benjamin Vermeer¹, Dong Hoon Shin^2,3, Alexander Vogel⁴, Fabian Zundel¹, Sabina Caneva², and •Sonja Schmid^1,4 — ¹University of Basel, Basel, Switzerland — ²Delft University of Technology, Delft, The Netherlands. — ³Korea University, Sejong, Republic of Korea — ⁴Swiss Nanoscience Institute, Basel, Switzerland

Paradoxically, single-molecule FRET studies rely on ensemble averaging during data analysis, because early photo-bleaching prohibits sufficient sampling of single molecules. As a result, the FRET-based study of inter- and intra-molecular heterogeneity in biomolecular function - a specific hallmark of single-molecule techniques - is hardly possible, preventing insights into dynamic disorder, the effects of post-translational and other modifications, of rare but decisive states, etc. Here, we demonstrate hour-long single-molecule FRET observations using DyeCycling in zero-mode waveguides, which circumvents photobleaching through reversible fluorophore binding. We detect the conformational dynamics of single molecules over four orders of magnitude in time (milliseconds-hour), enabling us to directly observe slow kinetic regime changes within individual molecules that were intractable previously. Moreover, we demonstrate the versatility of DyeCycling with DNA and protein molecules. Together, these advances establish DyeCycling/FRET as a powerful new approach that vastly expands the information gain of single-molecule FRET, enabling the study of important biological questions that were previously inaccessible.

Keywords: Single-molecule FRET; conformational change; dynamics; heterogeneity; dynamic disorder